Description

Multiplex PCR can simultaneously detect two or more products in a single reaction. There is an increasing demand for multiplex PCR techniques in assays conducted in research laboratories and forensic/diagnostic genotyping assays (1,2). Multiplex PCR can also be used for semi-quantitative gene expression analysis using cDNA templates.
The NEB Multiplex PCR 5X Master Mix is an easy-to-use solution featuring high quality recombinant Taq DNA Polymerase. The mix is optimized for high yield and robust performance. Its performance is illustrated in a 15-plex PCR using human genomic DNA (Figure 1) and an 8-plex PCR using cDNA products as templates (Figure 2). The 5X formulation allows the user maximal input of primers, template DNAs and additional components.

Figure 115-plex PCR using varying amounts of human genomic DNA. 1X Multiplex PCR 5X Master Mix was used with 0.15 μM of each primer. The cycling conditions were 95°C for 1 minute, 35 cycles of 95°C for 20 seconds, 60°C for 1 minute and 68°C for 2 minutes. Marker M is the 2-Log DNA Ladder (NEB #N3200).Figure 28-plex PCR using cDNA products from 1 ng human spleen total RNA. Cycling conditions were 95°C for 1 minute, 30 cycles of 95°C for 20 seconds, 60°C for 30 seconds and 68°C for 2 minutes. Amplicon lengths are indicated next to gel.Figure 3Comparison of PCR product yields between single-plex and multiplex PCR reactions. Figure A shows amplification of human genomic DNA fragments (sizes are listed to the left of the gel). Lane 1 is a 3-plex reaction, Lanes 2-4 are single-plex reactions, and Lanes 5–6 are 2-plex reactions; Lane 7 is the 1 kb DNA Ladder (NEB #N3232 ). Figure B shows the results of expression analysis of five mRNAs using cDNA templates (first strand synthesis was carried out using the ProtoScript Kit with human spleen total RNA); Lane 1 is a 5-plex PCR from cDNA reactions, while Lanes 2–6 are single-plex PCR reactions; Lane 7 is the 100 bp DNA Ladder (NEB #N3231).

Tech Tips

Test each primer set individually before combining into a multiplex reaction. The concentration of some primer pairs may need to be adjusted to achieve consistent and robust amplification across all amplicons in the mix.

Protocols

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Troubleshooting Guides

Interactive Tools

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Legal and Disclaimers

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Trademarks

NEW ENGLAND BIOLABS® and THERMOPOL® are registered trademarks of New England Biolabs, Inc.

IGEPAL® is a registered trademark of Rhodia Operations.TWEEN® is a registered trademark of Uniqema Americas, LLC.

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